Azhikina T, Kostina M, Skaptsova N, Potapov V, Berg D, Sverdlov E
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia.
DNA Seq. 1996;6(4):211-6. doi: 10.3109/10425179609008445.
We use modified oligonucleotides with enhanced strength of complementary DNA binding for primer walking DNA sequencing with strings of short contiguous oligonucleotides as primers. Such an approach allows us to reduce the probability of primer failures due to unstable binding of oligos with templates. In this paper the factors affecting the priming efficiency of segmented primers (strings composed of several short oligonucleotides contiguously juxtaposed on the template) used for DNA sequencing were investigated. Modified oligonucleotides were used to discriminate the effects caused by intrinsic properties of the oligonucleotides and by template features. It was shown that the most crucial factor is the stability of the duplex formed by the template with the 3'-outermost oligonucleotide of a string. The data were obtained with a model M13 template and in the process of sequencing the region flanking a long terminal repeat of human endogenous retrovirus HERVK mapped on chromosome 19. The sequencing was done by primer walking with strings of contiguous modified hexanucleotides. The effects revealed should be taken into consideration when choosing oligonucleotide units of segmented primers and for construction of minimised libraries composed of short unit oligonucleotides.
我们使用具有增强互补DNA结合强度的修饰寡核苷酸，以短串联寡核苷酸串作为引物进行引物步移DNA测序。这种方法使我们能够降低由于寡核苷酸与模板不稳定结合而导致引物失败的概率。本文研究了影响用于DNA测序的分段引物（由几个短寡核苷酸在模板上连续并列组成的串）引发效率的因素。使用修饰寡核苷酸来区分由寡核苷酸的内在特性和模板特征引起的影响。结果表明，最关键的因素是模板与串的3'-最外侧寡核苷酸形成的双链体的稳定性。这些数据是通过模型M13模板获得的，并且是在对位于19号染色体上的人类内源性逆转录病毒HERVK的长末端重复序列侧翼区域进行测序的过程中获得的。测序是通过用连续的修饰六核苷酸串进行引物步移来完成的。在选择分段引物的寡核苷酸单元以及构建由短单元寡核苷酸组成的最小化文库时，应考虑所揭示的影响。
